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Aquaculture 252 (2006) 79 84

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Screening of cultivated seaweeds for antibacterial activity against


fish pathogenic bacteria
Anne Bansemir, Maja Blume, Susanne Schrder, Ulrike Lindequist
Institute of Pharmacy, Department of Pharmaceutical Biology, Ernst-Moritz-Arndt University Greifswald, Friedrich-Ludwig-Jahn-Strasse 17,
D-17487 Greifswald, Germany
Received 6 April 2005; accepted 18 November 2005

Abstract
Because of the evolving resistance of microorganisms to existing antibiotics, there is an increasing need for new antibiotics not
only in human but also in veterinary medicine. Competition for space and nutrients led to the evolution of antimicrobial defence
strategies in the aquatic environment. Therefore, aquatic organisms, e.g., seaweeds, offer a particularly rich source of potential new
drugs. The aim of our studies was to identify seaweeds, which possess activities against fish pathogenic bacteria and could be an
alternative to the commonly used antibiotics in aquaculture.
Dichloromethane, methanole and water extracts of 26 species of cultivated seaweeds were screened for their antibacterial
activities against five fish pathogenic bacteria strains (Aeromonas salmonicida ssp. salmonicida, Aeromonas hydrophila ssp.
hydrophila, Pseudomonas anguilliseptica, Vibrio anguillarum, Yersinia ruckeri). The dichloromethane extracts of Asparagopsis
armata, Ceramium rubrum, Drachiella minuta, Falkenbergia rufolanosa, Gracilaria cornea and Halopitys incurvus showed strong
antibacterial activities. V. anguillarum and P. anguilliseptica were the two most susceptible bacteria strains. The screening results
confirm the possible use of seaweeds as a source of antimicrobial compounds or as a health-promoting food for aquaculture.
2005 Elsevier B.V. All rights reserved.
Keywords: Algae; Screening; Antimicrobial activity; Fish pathogenic bacteria

1. Introduction
Antibiotic treatment of bacterial diseases in fish
culture has been applied for many years. The occurrence
of antibiotic resistant bacteria associated with fish
diseases is a worldwide problem in aquaculture, which
has received considerable attention in the last years and
continues to increase due to the absence of a more
effective and safer use of antibiotics. The prevention and
treatment of these infectious diseases by applying
Corresponding author. Tel.: +49 3834 86 4868; fax: +49 3834 86
4885.
E-mail address: lindequi@uni-greifswald.de (U. Lindequist).
0044-8486/$ - see front matter 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2005.11.051

products from marine organisms appears as a possible


alternative. Hence, the interest in marine organisms as a
potential and promising source of pharmaceutical agents
has increased during the last years (Lindequist and
Schweder, 2001; Mayer and Hamann, 2002; Newman et
al., 2003). Seaweeds are considered as such a source of
bioactive compounds as they are able to produce a great
variety of secondary metabolites characterized by a
broad spectrum of biological activities. Compounds
with cytostatic, antiviral, anthelmintic, antifungal, and
antibacterial activities have been detected in green,
brown and red algae (Lindequist and Schweder, 2001;
Newman et al., 2003). There are numerous reports
concerning the inhibiting activities from macroalgae

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A. Bansemir et al. / Aquaculture 252 (2006) 7984

Table 1
Classification and origin of studied algae

Table 1 (continued)

Species

Class

Location

Origin

Anotrichum
furcellatum
(J. Agardh)
Baldock
Asparagopsis
armata Harvey
Ceramium rubrum
(Hudson)
C. Agardh
Chondrus crispus
Turner
Codium taylorii
P.C. Silva
Corallina elongata
J. Ellis and
Solander
Cystoseira
abies-marina
(Turn.) Agardh
Dictyota
dichotoma
(Hudson)
J.V. Lamouroux
Drachiella minuta
(Kylin)
Maggs and
Hommersand
Enteromorpha
compressa
(L.) Nees
Falkenbergia
rufolanosa
(Harvey)
F. Schmitz
Fryeella gardneri
(Setchell) Kylin
Gracilaria bursapastoris Gmelin
Gracilaria cornea
J. Agardh
(red variant: r)
Gracilaria cornea
J. Agardh
(green
variant: g)
Gracilaria cornea
J. Agardh (m)
Gracilaria
verrucosa
(Hudson)
Papenfuss
Grateloupia
dichotoma
J. Agardh
Grateloupia
doryphora
(Montagne)
M.A. Howe

Rhodophyceae

Atlantic, Faro,
Portugal

Rhodophyceae
Rhodophyceae

Rhodophyceae
Chlorophyceae
Rhodophyceae

Atlantic, Pleubian,
France
North Sea,
Helgoland,
Germany
Atlantic, Mindelo
Beach, Portugal
Atlantic, East
coast Gran Canaria
Atlantic, East
coast Gran Canaria

3*
1*

2
5
5

Phaeophyceae

Atlantic, East
coast Gran Canaria

Phaeophyceae

Atlantic, Faro,
Portugal

Rhodophyceae

Chlorophyceae

Rhodophyceae

Rhodophyceae
Rhodophyceae
Rhodophyceae

Atlantic, East
coast Gran Canaria

Atlantic, East
coast Gran Canaria

Atlantic,
Villefrance, France

Pacific, Fryday
harbour, Canada
Atlantiv, Aveiro
Lagoon, Portugal
Carribean Sea

1
2
5

Species

Class

Location

Origin

Halopitys incurvus
(Hudson)
Batters
Hypnea spinella
(C. Agardh)
Ktzing
Hypoglossum
hypo-glossoides
(Stackhouse)
F.S.Collins and
Hervey
Laminaria
saccharina (L.)
J.V. Lamouroux
Laurencia
chondrioides
B?rgesen
Palmaria palmata
(L.) Kuntze
Plocamium
cartilagineum
(L.) P.S. Dixon
Ulva rigida
C. Agardh
Ulva rigida
C. Agardh
Valonia utricularis
(Roth)
C. Agardh

Rhodophyceae

Atlantic, East
coast Gran Canaria

Rhodophyceae

Atlantic, East
coast Gran Canaria

Rhodophyceae

North Sea,
Helgoland,
Germany

Phaeophyceae

North Sea, Sylt,


Germany

Rhodophyceae

Atlantic, East
coast Gran Canaria

Rhodophyceae

Atlantic, Roscoff,
France
North Sea,
Helgoland,
Germany
Atlantic, Faro,
Portugal
Atlantic, East
coast Gran Canaria
Atlantic, East
coast Gran Canaria

Rhodophyceae

Chlorophyceae
Chlorophyceae
Chlorophyceae

4
5
5

1Sylt, Germany; 2Porto, Portugal; 3Pleubian France; 4Faro,


Portugal; 5Gran Canaria, Spain.
*Collected from natural sources.

against human pathogens, fungi and yeasts, but only few


contain data about effects against fish pathogens
(Sridhar and Vidyavathi, 1991; Mahasneh et al., 1995;
de Val et al., 2001; Liao et al., 2003).
Therefore, the aim of the present study was to
investigate the antimicrobial activity of extracts of
marine algae against five fish pathogenic bacteria that
are often the cause of bacterial diseases in aquaculture.
The investigated seaweeds were selected by seaweed
specialists of the EU project SEAPURA according to
their potential as component of integrated aquaculture
systems, as sources of pharmaceuticals, fine chemicals
or cosmetics and as fish feed. The possible use of active
seaweeds for prevention or treatment of the bacterial
fish diseases should be discussed.

Rhodophyceae

By cultivation of
red variant

Rhodophyceae

Mixture of red and


green variant
Atlantic, East
coast Gran Canaria

Rhodophyceae

Atlantic, East
coast Gran Canaria

2. Materials and methods

Rhodophyceae

Atlantic, East
coast Gran Canaria

2.1. Algal materials and preparation of the material

Rhodophyceae

Seaweeds (19 Rhodophyceae, 3 Phaeophyceae, 4


Chlorophyceae) were collected or cultivated at five

A. Bansemir et al. / Aquaculture 252 (2006) 7984

different places of Europe (Sylt, Germany; Porto,


Portugal; Pleubian, France; Faro, Portugal; Gran
Canaria, Spain, Table 1). Asparagopsis armata was
collected at the Atlantic coast of France by the group of
CEVA, Pleubian (Dr. Patrick Dion). Ceramium rubrum
was collected in the North Sea by the group of the
Alfred-Wegner-Institut (Prof. Klaus Lning). All other
seaweeds were cultivated by the collaborating groups of
the EU project SEAPURA (Sylt: Stiftung AlfredWegner-Institut, Prof. Klaus Lning; Porto: Universidade de Porto, Instituto Botanico, Dr. Isabel Sousa
Pinto; Faro: Universidade do Algarve, Centre of Marine
Sciences, Prof. Rui Santos; Gran Canaria: Universidad
de Las Palmas de Gran Canaria, Instituto de Algologia
Aplicada, Prof. Guillermo Garcia Reina). Falkenbergia
rufulanosa is the tetrasporangial phase of the species A.
armata. Because both generations of this species were
investigated separatedly, A. armata and F. rufulanosa
amounted separatedly. From Gracilaria cornea 3
variants were used. The Ulva rigida samples came
from 2 different locations. The fresh samples were
washed with seawater and fresh water to remove salts,
epiphytes, microorganisms and other suspended materials. The clean algae were frozen and lyophilized. The
dry material was stored at 20 C. Voucher specimens
are deposited in the Institute of Pharmacy, Department
of Pharmaceutical Biology, Ernst-Moritz-Arndt-University Greifswald, Germany.
2.2. Preparation of seaweed extracts
Extracts of the freeze dried and powdered biomass
were prepared using different organic solvents with
increasing polarity (dichloromethane, methanole and
water). Each extraction was carried out in a Soxhlet
apparatus for 24 h and after evaporation in vacuum the
extracts were stored at 20 C until use.
2.3. Antibacterial tests
The agar diffusion assay was performed according to
European Pharmacopoe (1997). One loopful of each test
organism (Vibrio anguillarum DSMZ 11323, Pseudomonas anguilliseptica DSMZ 12111, Yersinia ruckeri
ATCC 29493, Aeromonas salmonicida sp. salmonicida
ATCC 51413, Aeromonas hydrophila sp. hydrophila
DSMZ 6173) was suspended in 3 ml 0.9% NaCl
solution separately. Nutrient agar (Difco Tryptic Soy
Agar, Becton Dickinson and Company, USA) was
inoculated with this suspension of the respective
organism and poured into a sterile petri dish. Sterilised
paper discs, containing the extract of the alga (2 mg),

81

were transferred onto these prepared petri dishes.


Oxytetracycline (Merck, Germany) was used as a
positive, the solvent of each extract as a negative
control. A pre-diffusion for 3 h was guaranteed.
Inhibition zones were measured after 18 h incubation
at 26 C. The inhibition zones were measured excepting
the 6 mm paper disc. After pre-diffusion and incubation
the petri dishes were sprayed with a colouring solution
(p-iodo nitrotetrazolium violet, 5% in 50% aqueous
ethanole). Living bacteria produce a red coloured
compound, the inhibition zone appears colourless
(Brantner, 1997). Every experiment was carried out 10
times. Inhibition zones N 15 mm were declared as strong,
from 8 to 15 mm as moderate and from 1 to 8 mm as
weak activities. Minimal inhibitory concentration (MIC)
values against V. anguillarum and P. anguilliseptica
were determined by standard serial broth microdilution
assay (European Pharmacopoe, 1997).
2.4. Statistical analysis
The data were statistically analysed by applying an
one-way ANOVA.
3. Results
The results of the antimicrobial screening assays are
summarized in Table 2. It can be seen that only the
dichloromethane extracts showed significant inhibitory
effects. Dichloromethane extracts of 6, 4 or 11 samples
exhibited strong, moderate or weak antimicrobial
activity, respectively. 8 dichloromethane extracts did
not display activity. Considering the methanole extracts,
only 6 species showed moderate or weak antimicrobial
activity, and no water extract exhibited antibacterial
activity.
The two most susceptible organisms were V.
anguillarum and P. anguilliseptica, which were inhibited by extracts of 17 and 15 species, respectively. The
strongest antibacterial activities were obtained by A.
armata with inhibition zones of 19 mm against V.
anguillarum. Considerable antimicrobial activities
against V. anguillarum were presented also by Falkenbergia rufolanosa and G. cornea (r, g, m). The MIC
values against V. anguillarum were b 100 g/ml for A.
armata and b 400 g/ml for C. rubrum, F. rufolanosa,
G. cornea and Halopitys incurvus. The MIC values of
the other extracts were N 400 g/ml. The MIC value for
oxytetracycline against V. anguillarum amounted to
0.5 g/ml.
The growth of P. anguilliseptica was strongly
inhibited by A. armata and G. cornea (g) (27 mm).

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A. Bansemir et al. / Aquaculture 252 (2006) 7984

Table 2
Antimicrobial activity of the investigated dichloromethane-extracts (2 mg/disc) of seaweed species in agar diffusion assay (inhibition zone was
measured without paper disc)
Species

Anotrichum furcellatum
Asparagopsis armata
Ceramium rubrum
Chondrus crispus
Codium taylorii
Corallina elongata
Cystoseira abies-marina
Dictyota dichotoma
Drachiella minuta
Enteromorpha compressa
Falkenbergia rufolanosa
Fryeella gardneri
Gracilaria bursa-pastoris
Gracilaria cornea (r)
Gracilaria cornea (g)
Gracilaria cornea (m)
Gracilaria verrucosa
Grateloupia dichotoma
Grateloupia doryphora
Halopitys incurvus
Hypnea spinella
Hypoglossum
hypoglossoides
Laminaria saccharina
Laurencia chondrioides
Palmaria palmata
Plocamium cartilagineum
Ulva rigida
Ulva rigida
Valonia utricularis
Oxytetracycline 20 g/disc

Inhibition zones (mm,) against


Vibrio anguillarum Pseudomonas
anguilliseptica

Aeromonas
salmonicida

Aeromonas
hydrophila

Yersinia ruckeri

5.6 2.2
19.3 1.3
6.1 1.5
1.4 0.4
0
0
5.0 2.0
5.1 2.7
1.4 1.4
0
14.5 1.7
0
4.9 2.8
10.1 2.2
13.1 2.0
12.4 3.6
2.2 1.5
0.6 1.8
0
6.3 2.2
0
7.6 4.1

2.5 2.3
26.9 2.2
17.0 3.8
0
0.4 0.5
0
2.1 1.4
1.6 0.9
15.0 3.7
3.1 2.3
15.1 1.0
0
1.5 1.0
4.1 2.9
27.2 9.9
5.1 1.7
0
0.7 1.2
0
17.2 3.9
0
3.1 3.6

3.7 2.0
17.0 2.2
2.6 3.4
0
0
0
0
0
0
0
7.6 1.7
0
0
0.3 0.6
3.3 1.8
1.1 0.8
2.4 1.7
0.3 0.9
0
5.4 4.4
0
0

0.4 0.7
14.9 2.7
0
0
0
0
0
0
0
0
12.5 0.8
0
0
0
2.5 1.2
0
0
0
0
12.5 1.6
0
0

0
15.3 1.7
0
0
0
0
0
0
0
0
7.3 1.9
0
0
0
3.0 2.6
0
0
0
0
8.9 3.0
0
0

3.7 1.9
6.9 2.2
0
2.8 1.2
2.3 1.8
3.0 1.4
0
20.0

0
8.8 7.3
0
6.3 1.2
0.8 1.1
3.1 0.7
0

0
0
0
2.8 1.9
0
0
0
27.0

0
0.4 1.2
0
0.2 0.6
0
0
0
31.0

0
0
0
0
0
0
0
14.5

Inhibition zones N 15 mm were declared as strong (bold), from 8 to 15 mm as moderate and from 1 to 8 mm as weak activities.

Inhibition zones between 14 mm and 17 mm were


measured for C. rubrum, Drachiella minuta, F. rufolanosa and H. incurvus. MIC values b 400 g/ml for P.
anguilliseptica were only attained by G. cornea (g) and
F. rufolanosa extracts. The MIC value for oxytetracycline against P. anguilliseptica was 0.08 g/ml.
An influence on the growth of Aeromonas salmonicida was detected for 8 seaweed species. Asparagosis
armata showed the strongest activity (17 mm). H.
incurvus and F. rufolanosa developed inhibition zones
between 5 and 10 mm.
Only 3 extracts (A. armata, H. incurvus, F.
rufolanosa) showed antimicrobial activities against
Aeromonas hydrophila that are worth mentioning.
Antimicrobial effects against Y. ruckeri were presented by A. armata, F. rufolanosa and H. incurvus.
Among the methanole extracts only those of C.
rubrum, F. rufolanosa and H. incurvus demonstrated

weak inhibiting activities against V. anguillarum, P.


anguilliseptica and A. salmonicida.
Summarizing the results it can be conducted that the
most effective seaweeds were A. armata, C. rubrum, D.
minuta, F. rufolanosa, G. cornea (g) and H. incurvus.
4. Discussion
The main objective of this study was to evaluate
the ability of different cultivated seaweed species
from several location to inhibit the growth of fish
pathogenic bacteria with the aim to use them in the
future as alternatives to common antibiotics in
aquaculture.
Most active species were A. armata, C. rubrum, D.
minuta, F. rufolanosa, G. cornea and H. incurvus. They
belong to the Rhodophyceae. The high efficiency of
seaweeds belonging to the Rhodophyceae agrees with

A. Bansemir et al. / Aquaculture 252 (2006) 7984

the results of previous studies using other test microorganisms (Mahasneh et al., 1995; Padmakumar and
Ayyakkannu, 1997). The MIC values of the extracts are
much higher than those of the positive control substance
oxytetracycline. This is not surprisingly because extracts
are complex mixtures of many compounds and the
portion of active compounds is very low.
Several solvents were used for the extraction of
freeze-dried seaweed powder. Our data revealed that
considerable inhibition zones were only observed for
the dichloromethane extracts. Therefore, the compounds responsible for the antimicrobial activity are
lipophilic. We assume, that the active compounds
could be, at least partly, lipophilic halogenated
compounds. Halogen-containing terpenoids, acetylens
and phenols have been identified in several seaweed
species as biologically active compounds (Knig and
Wright, 1997; Carvalho and Roque, 2000; Vairappan
et al., 2001). The two most active dichloromethane
extracts were those of A. armata and F. rufolanosa.
Both algae represent different generations of the same
species of red algae. Whereas A. armata represents the
gametangial phase, F. rufolanosa is the tetrasporangial
phase. They are morphological dissimilar (Knappe,
1980). A series of small molecular volatile halogenated compounds (halomethanes, haloether, haloacetales)
accounts for the antimicrobial action of A. armata
(McConnell and Fenical, 1977). They could also be
responsible for the observed effects against fish
pathogenic bacteria. Considering Laurencia chondrioides, possessing moderate activity, we found two
halogenated sesquiterpenes as responsible metabolites
for the observed activity against fish pathogenic
bacteria (Bansemir et al., 2004). Besides halogenated
compounds, fatty acids have been identified as
antimicrobial substances in algae (Rosell and Srivastava, 1987). We showed that C. rubrum contains
several fatty acids with antimicrobial activities (Bansemir, 2004). The structure elucidation of further
active compounds is in progress.
Some halogenated compounds from algae possess
cytotoxic and mutagenic properties (Teuscher and
Lindequist, 1994). Before starting the use of seaweeds
for prophylaxis and therapy of bacterial fish diseases in
vitro and in vivo toxicity studies with seaweeds,
fractions and purified compounds have to be done.
Besides, investigations about the stability of the
materials in an aquatic environment, about digestability
for fish and about the metabolism of the active
compounds in fishes would be necessary. Only
dependent on these results a decision can be made
which material would be the most suitable for a possible

83

practical use. Toxicological tests using fish and human


cell cultures and investigations for digestability in fishes
are currently undertaken.
5. Conclusions
The results clearly show that seaweeds are an
interesting source for biologically active compounds
that may be applied for prophylaxis and therapy of
bacterial fish diseases additionally or instead of
commercial antibiotics. An alternative approach to the
use of extracts, fractions or purified compounds from
algae as drugs might be to employ seaweeds as fish feed
components. However, prior further investigations
regarding toxicity, stability and metabolism of seaweeds
and seaweed components must be undertaken.
Acknowledgement
This work is supported by EU grant Q5RS-200031334 (SEAPURA) in the specific programme Quality
of Life and Management of Living Resources.
Supply of algae biomass by our SEAPURA project
partners is gratefully acknowledged.

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